Bottom Line:
Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons.Finally, we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons.Thus, we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons.

BIO010447F2: Neurospheres can be generated from the hypothalamus of adult zebrafish. (A) Neural progenitors were disaggregated (black cells), plated, and maintained in proliferation media for 7 day (A1 to A3 neurospheres, −7 to 0 days). Cells were then seeded (A4 to A6, upper panel) on coated chamber wells and changed to differentiation media for 7 days (0 to +7 days). Alternatively, neurospheres (A3) were disaggregated and seeded (A4 to A6, bottom panel) on coated chamber wells and changed to differentiation media for 7 days with or without hormonal treatment (0 to +7 days). (B-D) 7 days in vitro bright field images of neurospheres (A3). Scale bar 30 μm.

Mentions:
To explore the possibility of GnRH neurogenesis in the adult zebrafish we developed a method to obtain neural progenitors from the brain of adult zebrafish. We dissected the region of the hypothalamus from fish aged one to two years and dissociated the cells mechanically to select individual cells for seeding (Fig. 2A1, black cells: progenitors). The resulting primary neurospheres, formed over two days, were mechanically disaggregated to individual cells (Fig. 2A2, white cells: progenitors slightly differentiated) and then seeded in cell culture chambers for 5 days resulting in undifferentiated secondary neurospheres (Fig. 2A3,B-D).Fig. 2.

BIO010447F2: Neurospheres can be generated from the hypothalamus of adult zebrafish. (A) Neural progenitors were disaggregated (black cells), plated, and maintained in proliferation media for 7 day (A1 to A3 neurospheres, −7 to 0 days). Cells were then seeded (A4 to A6, upper panel) on coated chamber wells and changed to differentiation media for 7 days (0 to +7 days). Alternatively, neurospheres (A3) were disaggregated and seeded (A4 to A6, bottom panel) on coated chamber wells and changed to differentiation media for 7 days with or without hormonal treatment (0 to +7 days). (B-D) 7 days in vitro bright field images of neurospheres (A3). Scale bar 30 μm.

Mentions:
To explore the possibility of GnRH neurogenesis in the adult zebrafish we developed a method to obtain neural progenitors from the brain of adult zebrafish. We dissected the region of the hypothalamus from fish aged one to two years and dissociated the cells mechanically to select individual cells for seeding (Fig. 2A1, black cells: progenitors). The resulting primary neurospheres, formed over two days, were mechanically disaggregated to individual cells (Fig. 2A2, white cells: progenitors slightly differentiated) and then seeded in cell culture chambers for 5 days resulting in undifferentiated secondary neurospheres (Fig. 2A3,B-D).Fig. 2.

Bottom Line:
Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons.Finally, we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons.Thus, we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons.